RESUMO
We have recently reported that cultured human monocytes are susceptible to lysis by autologous lymphokine-activated killer (LAK) cells. In an attempt to modulate the sensitivity of monocytes to LAK-mediated lysis, monocytes were cultured in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF was found to enhance the susceptibility of monocytes to lysis by LAK cells by 2- to 5-fold over that of untreated cells in a dose-dependent manner. As little as 10 units of GM-CSF per milliliter was sufficient to induce increased sensitivity. In a kinetics study, susceptibility of monocytes increased after 2 days of incubation with GM-CSF, with peak sensitivity occurring from 4 to 6 days of culture. The effect of GM-CSF appeared to be specific for monocytes within the circulating peripheral blood cells because nonadherent cells (NAC) and granulocytes, which are normally resistant to LAK-mediated lysis, did not become susceptible after treatment with GM-CSF. In cold-target inhibition experiments, unlabeled GM-CSF-treated monocytes, but not untreated monocytes, could block the lysis of FMEX, a human melanoma tumor cell line, as well as freshly isolated tumor cells. Finally, LAK cells specifically bound to GM-CSF-treated monocytes in significantly higher percentages than to control monocytes. In summary, our results indicate that GM-CSF was capable of enhancing the susceptibility of monocytes to LAK lysis possibly via increased binding or expression of target structure(s).
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Células Matadoras Naturais/imunologia , Linfocinas/farmacologia , Monócitos/imunologia , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Granulócitos/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Cinética , Linfócitos/citologia , Monócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
Using ascitic fluid or pleural effusion obtained from 13 ovarian or metastatic breast cancer patients, we separated tumor cells from effusion-associated lymphocytes (EAL) with Percoll density centrifugation. Lymphocytes were incubated with recombinant interleukin 2 (IL-2) for 3-4 days and then assessed for tumoricidal activity in a 51chromium-release assay. The IL-2-activated EAL were found to lyse autologous fresh tumor cells, as well as allogeneic fresh tumor cells and FMEX tumor cells, a melanoma cell line which is resistant to natural killer cell activity but is sensitive to lysis by lymphokine-activated killer cells. There was little or no tumoricidal activity seen in freshly isolated EAL or in EAL which were cultured in medium without IL-2. Phenotypically, the IL-2-activated EAL were largely CD3-, although some cytolytic activity was found in CD3+ populations. Also, most activity was found in cells positive for CD2 (OKT11) and CD16 (Leu 11b), and negative for the monocyte marker Leu M3. These results indicate that the activated cell types found in EAL were predominantly natural killer/lymphokine-activated killer-like with a small contribution from T-cells. Finally, EAL were readily activated by IL-2 in medium containing autologous effusion fluid, indicating that in situ activation of tumoricidal activity by IL-2 can occur in the face of potentially inhibitory substances or cells that may exist in the effusions. Direct introduction of IL-2 may therefore be a potential therapeutic modality of effusion-forming cancers.
Assuntos
Líquido Ascítico/imunologia , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias/imunologia , Derrame Pleural/imunologia , Adulto , Idoso , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3 , Fracionamento Celular , Citotoxicidade Imunológica , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
We have previously reported that cultured human monocytes are lysed by autologous lymphokine-activated killer (LAK) cells in vitro and that treatment of monocytes with interferon-gamma (IFN-gamma) decreased their sensitivity to lysis. Conversely, incubation of monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhanced their susceptibility to LAK-mediated cytotoxicity. To determine if certain antigens were differentially modulated on macrophages by IFN-gamma and GM-CSF, cytokine-treated and untreated monocytes were analyzed for the expression of a variety of cell surface markers by flow cytometry. Cytotoxicity assays were performed to assess the ability of antibodies to each of these markers to block LAK lysis of macrophage target cells. While several of the surface structures were differentially modulated by cytokine treatment, it was found that only monoclonal antibodies to the adhesion proteins CD11a and CD18 were capable of blocking lysis of either cytokine-treated or untreated target macrophages.
Assuntos
Antígenos de Superfície/análise , Citotoxicidade Imunológica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/farmacologia , Células Matadoras Ativadas por Linfocina/imunologia , Macrófagos/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Antígenos CD/fisiologia , Citometria de Fluxo , Humanos , Macrófagos/imunologiaRESUMO
We have previously reported that Legionella pneumophila antigens can induce interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in vitro and in vivo in mice. Furthermore, treatment of murine polymorphonuclear leukocyte (PMN) cultures with these cytokines resulted in augmented killing of the bacteria in vitro. The purpose of the present study was to determine if these findings could be extended to human responses. Here we report that Legionella antigens induced IFN-gamma and TNF in nonimmune human leukocytes cultures, and that these cytokines were able to stimulate the bactericidal activity of isolated PMN against L. pneumophila in vitro. Furthermore, optimal production of IFN-gamma was found in cultures which were enriched for large granular lymphocytes (LGL). The phenotype of IFN-producing cells was determined to be CD11+, CD16+, CD2+, and negative for CD4, CD8, CD14, and Leu 7. Additionally, Legionella-infected monocytes were found to produce TNF in a dose-dependent response to the number of infecting bacteria, and the addition of recombinant IFN-gamma to infected monocytes resulted in augmented production of TNF in a synergistic manner. Finally, treatment of PMN with recombinant IFN-gamma and recombinant TNF augmented their bactericidal activity against Legionella in a dose-dependent response. Thus, cytokines which can be induced by L. pneumophila antigens are able to stimulate PMN function in vitro, suggesting that resistance to infection results from a complex interaction of cytokines and cell responses.
Assuntos
Regulação da Expressão Gênica , Interferon gama/genética , Legionella/fisiologia , Neutrófilos/fisiologia , Fator de Necrose Tumoral alfa/genética , Anticorpos/imunologia , Citotoxicidade Imunológica , Humanos , Interferon gama/farmacologia , Doença dos Legionários/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fenótipo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This study shows that normal human large granular lymphocytes (LGL) secrete tumor necrosis factor (TNF) in response to Mycobacterium avium-intracellulare complex (MAI). Percoll density gradient fractionation of peripheral mononuclear cells showed TNF activity in the fractions corresponding to LGL and not T cells, even when 5% monocytes were added to the T lymphocytes for accessory function. TNF release was not abrogated by treatment of the crude LGL preparations with anti-Leu M3, -CD4, and -CD8 antibodies (Ab) plus complement (C), but was abrogated by anti-CD16 and -CD2 Ab, as expected. Interestingly, anti-HLA-DR monoclonal antibody (mAb) treatment significantly diminished TNF activity from LGL, but maintained natural killer (NK) cell function unmodified as opposed to CD2+ and CD16+ cell depletion. Panning studies demonstrated that TNF secretion upon MAI stimulation resided only in the HLA-DR+ LGL and not the DR- LGL population. These results indicate that normal fresh HLA-DR+ LGL, as well as monocytes, are also responsible for rapid TNF secretion during early MAI infection. These DR+ cells appear to be distinct from those expressing NK function.
Assuntos
Subpopulações de Linfócitos/imunologia , Complexo Mycobacterium avium/imunologia , Infecção por Mycobacterium avium-intracellulare/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Antígenos de Diferenciação/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos CD2 , Separação Celular , Antígenos HLA-DR/imunologia , Humanos , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Monócitos/imunologia , Receptores Fc/análise , Receptores de IgG , Receptores Imunológicos/análise , Fatores de TempoRESUMO
Tumor necrosis factor (TNF) was found in the lung lavage fluids of Legionella pneumophila-infected mice within 24 hr of intratracheal (i.t.) inoculation. Since this cytokine has been reported to activate polymorphonuclear leukocyte (PMN) function, the effect of TNF on the in vitro bactericidal capacity of PMN-enriched cultures was determined. Murine thioglycollate-elicited PMN which were treated with recombinant human TNF demonstrated augmented killing of L. pneumophila bacteria in vitro. Furthermore, treatment of PMN suspensions with cytokine-containing lung lavage fluid was found to enhance the bactericidal activity of PMN. The addition of anti-cachectin/TNF antibodies partially abrogated the stimulatory effects of the lavage fluid, suggesting that in vivo activation of PMN during the course of infection was likely, and that TNF was partially responsible for the enhanced bactericidal activity. In vivo treatment of animals with TNF resulted in significant protection of the animals from mortality. Furthermore, the rate of clearance of bacteria from the lung tissues of infected mice was increased in those animals treated with TNF, and correlated with the ability of this cytokine to protect the animals. These data suggest that the induction of TNF by Legionella bacteria during infection are involved in the non-specific host defense mechanisms, and that PMN activated by the TNF may be instrumental in clearing the organism from infected lung tissues, thereby protecting the animal.
Assuntos
Doença dos Legionários/prevenção & controle , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Líquido da Lavagem Broncoalveolar , Relação Dose-Resposta a Droga , Feminino , Doença dos Legionários/imunologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos , Neutrófilos/imunologiaRESUMO
[3H]glycerol was used to radiolabel Mycobacterium avium (MA) bacteria after interaction with human monocytes in a rapid in vitro assay for determination of the growth inhibition of the mycobacteria by monocytes. Monocytes and MA were co-cultured in 96-well microtiter plates for 1-5 days, and [3H]glycerol was added for an additional 3 days of incubation to radiolabel residual bacteria. The results indicate that monocytes inhibited mycobacterial growth within 24 h of co-culture, an activity which increased during incubation until optimal growth inhibition was noted by 3-4 days. A comparison with conventional plate counting methodology demonstrated similar responses between the two assays except that the conventional assay required 2-3 weeks of culture before visible MA colonies could be detected and enumerated. Thus, the development of a rapid radiolabel assay to quantitate the interaction between monocytes and MA will facilitate the investigation of normal host responses to this opportunistic pathogen.
Assuntos
Glicerol , Marcação por Isótopo/métodos , Monócitos/microbiologia , Mycobacterium avium/crescimento & desenvolvimento , Trítio , Contagem de Colônia Microbiana , Humanos , Mycobacterium avium/isolamento & purificação , Contagem de CintilaçãoRESUMO
Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that inactivate intracellular cyclic AMP (cAMP). Because the functions of T-lymphocytes are modulated by cAMP levels, the isozymes of PDE in these cells are potential targets for new drugs designed to modify the body's immunity through selective alteration of T-lymphocyte PDE activity. Cyclic GMP and 3(2H)-pyridazinone-4,5- dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-5-methyl-monohydrochloride (CI-930) selectively inhibit the catalytic activity of one of the two high affinity cAMP-PDE isozyme families known to occur in mammals, whereas d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone (Ro 20-1724) selectively inhibits the other. The objectives of this investigation were: (1) to determine whether human T-lymphocytes contain one or both of these pharmacologically distinguishable high-affinity cAMP-PDEs, and (2) to determine the effects of selective inhibitors of these PDEs on lymphocyte blastogenesis. High-affinity cAMP-PDE was found in both the soluble and particulate fractions of T-lymphocyte sonicates. Cyclic GMP and CI-930 inhibited PDE in the particulate fraction better than in the soluble fraction, but the converse was found for Ro 20-1724. CI-930 or Ro 20-1724, used alone, attenuated T-lymphocyte blastogenesis, but neither suppressed it completely. In combination, the same PDE inhibitors caused greater suppression of blastogenesis than either produced alone. The results indicate that human T-lymphocytes contain both CI-930- and Ro 20-1724-inhibitable isozymes. Either of the isozymes can modulate human T-lymphocyte blastogenesis, but inhibition of both isozymes produces synergistic antiblastogenic effects.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/sangue , Isoenzimas/sangue , Linfócitos T/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Fracionamento Celular , GMP Cíclico/farmacologia , Citometria de Fluxo , Humanos , Isoenzimas/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Papaverina/farmacologia , Piridazinas/farmacologia , Linfócitos T/citologiaRESUMO
Women at high risk for the development of breast cancer have several options open to them including increased cancer surveillance, prophylactic mastectomy and/or oophorectomy, and chemoprevention. We consider high-risk women to be those with known BRCA mutations or a strong family history characterized by multiple relatives with breast cancer, early age at diagnosis, and in some families, ovarian cancer. We present existing data regarding prophylactic surgery for these women. Essentially, a woman at high risk for breast cancer may choose to undergo bilateral prophylactic mastectomy, with or without reconstruction. For patients who have a known breast cancer, contralateral mastectomy is also an option. Finally, for women in families with a strong incidence of ovarian cancer, prophylactic oophorectomy can be considered.
Assuntos
Neoplasias da Mama/cirurgia , Mastectomia/métodos , Síndromes Neoplásicas Hereditárias/cirurgia , Prevenção Primária/métodos , Idade de Início , Neoplasias da Mama/genética , Neoplasias da Mama/psicologia , Feminino , Genes BRCA1 , Genes BRCA2 , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Humanos , Mamoplastia/efeitos adversos , Mamoplastia/métodos , Mastectomia/efeitos adversos , Mastectomia/psicologia , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/cirurgia , Ovariectomia , Seleção de Pacientes , Linhagem , Fatores de RiscoRESUMO
OBJECTIVES: The ascites-associated lymphocytes in ovarian cancer have altered immunologic function, and cell-free ascitic fluid has immunomodulating properties. We determined (1) whether interleukin (IL)-2 could induce lymphokine-activated killer (LAK) activity in normal peripheral blood mononuclear cells (PBMC) cultured in ovarian cancer ascitic fluid, and (2) whether IL-12 could synergize with IL-2 to generate LAK activity in normal PBMC cultured in ascitic fluid. METHODS: Normal PBMC were cultured in control medium and in media consisting of 50% ascitic fluid (ascitic medium), with and without IL-2 and IL-12. Cell activation to assess LAK activity (cell lysis) was determined in a 51Cr-release assay with the tumor cell lines FMEX and SKOV3 as target cells. To determine a possible mechanism for any synergistic effect, the expression of perforin, a pore-forming protein, was determined by Northern blot analysis. RESULTS: Interleukin-2 alone could not induce LAK activity in normal PBMC cultured in 50% ascitic fluid for up to 3 days. Interleukin-12 did mediate some or minimal LAK activity after 1, 2, or 3 days of incubation in control medium or in 50% ascitic fluid. When IL-2 and IL-12 were used in combination, PBMC cultured for 3 days in 50% ascitic fluid had remarkably high lytic activity against FMEX and SKOV3 tumor cells. In some experiments, this cytotoxicity was greater than that in PBMC cultured in control medium with IL-2 and IL-12. Lower concentrations of IL-12 (1 U/mL) with IL-2 (100 U/mL) were as effective as, and often more effective than, higher doses of IL-12 with IL-2. Very low-dose IL-12 (0.01-0.03 U/mL) in combination with IL-2 also induced a range of cytotoxicities. Only the combination of IL-2 and IL-12 up-regulated expression of perforin mRNA in ascitic medium. CONCLUSIONS: The cytotoxicity responses of PBMC cultured in ascitic fluid in the presence of IL-2 and IL-12 are complex. Low-dose IL-2 and IL-12 can overcome the inhibitory property of ascitic fluid on LAK generation and can restore and enhance cytotoxic activity, possibly by reconstituting the expression of perforin. These findings may have therapeutic potential.
Assuntos
Ascite/imunologia , Citotoxicidade Imunológica , Interleucina-12/farmacologia , Interleucina-2/farmacologia , Células Matadoras Ativadas por Linfocina/imunologia , Neoplasias Ovarianas/imunologia , Transcrição Gênica/efeitos dos fármacos , Ascite/patologia , Células Cultivadas , Sinergismo Farmacológico , Feminino , Humanos , Células Matadoras Ativadas por Linfocina/efeitos dos fármacos , Melanoma , Glicoproteínas de Membrana/biossíntese , Neoplasias Ovarianas/patologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , RNA Mensageiro/biossíntese , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Tumorais CultivadasRESUMO
Advanced epithelial ovarian cancer has recently been identified by us to be associated with elevated serum and ascitic levels of the soluble Interleukin-2 receptor alpha (sIL-2R alpha). To determine the cellular source of sIL-2R alpha, the expression of IL-2R alpha was assessed at the mRNA and protein level in peripheral blood mononuclear cells (PBMC), in ovarian cancer ascitic cell infiltrates and in primary and metastatic epithelial ovarian cancer lesions by immunochemistry, by flow cytometric analysis and by in situ hybridization (ISH). Normal PBMC and the PBMC from ovarian cancer patients had a low or undetectable level of IL-2R alpha mRNA and of IL-2R alpha cell-surface protein expression. Flow cytometric analysis of the heterogeneous ascitic infiltrates revealed few cells positively expressing cell-surface IL-2R alpha. By immunocytochemistry, 1-2% of leukocytes in the ascitic infiltrates were IL-2R alpha+. Cytologically these IL-2R alpha+ cells were lymphocytes. Frozen sections of primary and metastatic ovarian cancer lesions showed sparse lymphocytic infiltration and very small numbers of these tumour infiltrating lymphocytes (TIL) were IL-2R alpha+. In situ hybridization demonstrated that although less than 2% of leukocytes in the ascitic infiltrate had detectable levels of IL-2R alpha mRNA, there was a wide range in the level of mRNA expression in these positive cells. The cells expressing IL-2R alpha mRNA had the cytologic characteristics of lymphocytes. Similarly, in the frozen sections of the solid tumours, there was a range in the level of IL-2R alpha mRNA expression in the few TIL that expressed IL-2R alpha. Importantly, ovarian cancer cells and mesothelial cells did not express IL-2R alpha mRNA or IL-2R alpha protein. Our observations lead us to conclude that lymphocytes are the main, if not the only, source of sIL-2R alpha in ovarian cancer patients. Although cells expressing IL-2R alpha were relatively few in number, as the source of the high levels of sIL-2R alpha, they may contribute to the immunosuppression of ascitic lymphocytes in advanced epithelial ovarian cancer.
Assuntos
Neoplasias Epiteliais e Glandulares/química , Neoplasias Ovarianas/química , RNA Mensageiro/análise , Receptores de Interleucina-2/genética , Ascite/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoquímica , Metástase Neoplásica , Receptores de Interleucina-2/análiseRESUMO
Mycobacterium avium-intracellulare (MAI) is an ubiquitous soil contaminant that rarely causes disseminated disease in adults, regardless of immunological status. In AIDS patients, however, this microorganism invades virtually every tissue and organ, and most conventional chemotherapeutic agents are usually ineffective against MAI. We report here that monocytes, in which MAI has established an intracellular parasitic stage, appear to be under the control of natural killer (NK) cells. Autologous large granular lymphocytes (LGL), purified from human peripheral blood mononuclear cells (PBMC), were capable of efficiently lysing MAI-infected monocytes in a 5 hr 51Cr-release assay. More importantly, interleukin 2 (IL-2) was able to activate the LGL to a high degree of lysis of infected monocytes. Additionally, 3 to 4 days of incubation of LGL with MAI resulted in the induction of killer cells capable of killing bacterially-infected monocytes, as well as tumor cells. Northern blot analysis of RNA from MAI-stimulated LGL revealed specific messages for both IL-2 receptor proteins (p55 and p70). Thus, MAI can directly activate killer cells, which may therefore play a role in containment of MAI infection by lysis of parasitized monocytes before the bacteria can multiply and spread to other sites.
Assuntos
Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Complexo Mycobacterium avium/imunologia , Citotoxicidade Imunológica , Humanos , Técnicas In Vitro , Linfócitos/imunologia , Monócitos/microbiologia , Receptores de Interleucina-2/metabolismoRESUMO
L. pneumophila is a facultative intracellular opportunistic pathogen ubiquitously present in the environment. Much is now known concerning the ecological niche of this organism as well as many other characteristics of these bacteria, including physiology and biochemistry. However, much less is known about immune mechanisms responsible for host resistance vs susceptibility. Not only outer membrane protein rich fractions but also LPS-rich components are potent immunogens, both in experimental animals such as susceptible guinea pigs and more resistant rodent species like rats and mice. Immunity to these organisms can be readily observed by a variety of serologic techniques. Antibody titers increase rapidly after exposure of individuals to these bacteria either by infection or immunization. However, such antibody does not appear to play an important role in host resistance. Serum antibody plus complement is not lytic for the bacteria in vitro. Furthermore, antibody appears to promote the phagocytosis of the bacteria by monocytes and/or macrophages in culture but such phagocytosis does not result in killing of the bacteria, merely an enhanced uptake and subsequent replication of the organisms. Studies on cellular immunity have focused attention on the role of T lymphocytes, monocytes and macrophages. In addition, cutaneous hypersensitivity is readily induced by infection or immunization of experimental animals with Legionella or antigenic components. In vitro correlates of hypersensitivity is also readily evident after infection or immunization. Although lymphoid cells from guinea pigs only show evidence of responsiveness to Legionella antigens by the lymphocyte blastogenic reaction after animals have been sensitized, peripheral blood monocytes from man as well as splenocytes from mice show evidence of responsiveness to Legionella even before known infection or sensitization. However, higher blastogenic responses become evident after sensitization or infection. In addition, interleukins, such as interleukin 1 and 2, as well as interferon and tumor necrotizing factor, appear in response to Legionella antigens and seem to play a role in resistance mechanisms. Cellular replication of Legionella in monocytes from man as well as macrophages from susceptible animals seems related to susceptibility or resistance to these organisms. Further analyses of the nature and mechanism of humoral vs cellular immune responses to Legionella antigens will provide valuable information about immunity and resistance to these intracellular pathogens in susceptible individuals.
Assuntos
Imunidade , Doença dos Legionários/imunologia , Animais , Humanos , Legionelose/imunologiaAssuntos
Infecções por Bacteroides/metabolismo , Endotoxinas/farmacologia , Lipopolissacarídeos/farmacologia , Animais , Infecções por Bacteroides/patologia , Bacteroides fragilis/metabolismo , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Endotoxinas/toxicidade , Escherichia coli/metabolismo , Feminino , Interferons/biossíntese , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Baço/citologia , Baço/metabolismo , Timidina/metabolismoRESUMO
Mycobacteria are acid-fast, slow-growing microorganisms which have gained attention due to increasing prevalence in AIDS patients. Until the advent of AIDS, the only true pathogens of this group were Mycobacterium tuberculosis and M. leprae and the remaining mycobacteria were considered to be saprophytes or opportunistic pathogens. Infection with the MOTT (mycobacteria other than tuberculosis) bacilli was only seen in elderly or immunocompromised patients and was generally limited to caseating pulmonary granulomas, with rare extrapulmonary involvement. In AIDS patients, however, the incidence of mycobacterial infections ranges from 10 to 60% of HIV-positive persons, depending on location, method of identification, and patient population. Furthermore the pathogenesis of these mycobacterioses is distinct from that seen in non-AIDS patients because disseminated disease is the rule rather than the exception. Finally treatment of mycobacterial infections is increasingly difficult due to multiple drug resistances as well as the length of antimicrobial therapy required to cure the disease. Because of the prevalence and importance of these microorganisms, much research has been performed with the mycobacteria to develop new therapies and to understand their modes of pathogenesis.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Infecções por Mycobacterium/complicações , Infecções por HIV/complicações , HumanosRESUMO
We have recently reported that IL 2-activated killer (LAK) cells are capable of lysing cultured human monocytes. In an effort to protect autologous monocytes from lysis, we treated monolayer cultures of adherent PBMC with various doses of human rIFN-gamma and assessed their susceptibility to LAK cells. IFN-gamma was shown to lessen the sensitivity of monocytes to lysis in a dose-dependent manner. Similar treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 h incubation with IFN-gamma was sufficient for the protective effects to take effect. Additionally, monocytes that were pulsed with IFN-gamma for 2 h, washed, and then cultured in medium alone retained their resistance to lysis for at least 3 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Furthermore, binding studies demonstrated that there was no significant difference between the number of conjugates formed by using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal. From these studies, it was apparent that treatment of monocytes with IFN-gamma lessened their sensitivity to LAK-mediated lysis. Thus, it may be possible through a specific sequence of IFN-gamma and IL-2 treatment that LAK activity could be manipulated against some tumor cells, but not normal cells, to abrogate some of the toxicity seen with this type of cancer therapy.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-2 , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Células Cultivadas , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/farmacologia , Monócitos/classificação , Monócitos/metabolismo , Fenótipo/efeitos dos fármacos , Biossíntese de Proteínas , RNA/biossíntese , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.
Assuntos
Citotoxicidade Imunológica , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Anticorpos Monoclonais , Antígenos de Diferenciação/análise , Separação Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ativação Linfocitária/efeitos dos fármacos , Povidona , Dióxido de SilícioRESUMO
Interleukin-2 (IL-2)-activated killer (LAK) cells specifically lyse human monocytes, which may account for some of the toxicity seen during LAK/IL-2 immunotherapy of cancer patients. In an effort to protect autologous monocytes, we treated monolayer cultures of monocytes with various doses of recombinant human interferon-gamma (IFN-gamma) and assessed their sensitivity to LAK-mediated lysis. IFN-gamma lessens the sensitivity of monocytes to lysis in a dose-dependent manner. Treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 hr incubation with IFN-gamma was sufficient for protection to occur, and that monocytes which were treated with IFN-gamma for 2 hr, washed, and then cultured in medium alone retained their resistance to lysis for at least 4 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Finally, binding studies demonstrated that there was no significant difference between the number of conjugates formed using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Monócitos/efeitos dos fármacos , Produtos Biológicos/farmacologia , Fatores Estimuladores de Colônias/farmacologia , Citocinas , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/farmacologia , Humanos , Indometacina/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Cinética , Monócitos/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Using a 24-hr radiolabel microassay developed in our laboratory that measures [3H]glucose uptake in residual Candida, we have identified the effector cells responsible for in vitro inhibition of Candida albicans growth as mainly polymorphonuclear neutrophils (PMN) and monocytes within the human peripheral blood cells. Highly purified T cells and large granular lymphocytes (LGL) that mediate natural killer activity which were obtained by Percoll density gradient centrifugation were found to have no innate activity against C. albicans. The LGL could not be activated by interferon-alpha, interferon-gamma or interleukin 2 to inhibit Candida growth although their K562 tumor cytotoxic activity was readily enhanced by these cytokines. Stimulation with heat-killed C. albicans also did not activate fungal growth inhibitory function in LGL and the supernatant of these activated LGL had no direct fungicidal activity. However, the activated LGL supernatant had the capability to enhance PMN function against C. albicans growth. Addition of recombinant human tumor necrosis factor, affinity-purified interferon-alpha, or interferon-gamma to PMN caused increased antifungal activity in PMN. However, antibodies to these cytokines had only a partial adverse effect on the ability of the activated LGL supernatant to stimulate PMN anti-Candida function. Therefore, the activated LGL supernatant appeared to contain a potent stimulator of PMN function which is as yet unidentified. These data indicate that LGL did not directly mediate anti-Candida activity but could indirectly influence C. albicans growth by activating PMN against the fungi through the release of a specific PMN-activating factor. Our findings therefore add another role to LGL which is the regulation of PMN function, the consequence of which is regulation of fungal immunity.